TWI457131B

TWI457131B - Peptides and use thereof in the inhibition of angiotensin converting enzyme

Info

Publication number: TWI457131B
Application number: TW101132603A
Authority: TW
Inventors: Yi Hong Chen; Hsiang Ling Lai; Shiao Cheng Chuang; Chien Ti Chang; Ming Yu Hung; yu hui Liu; Su Er Liou; Fu Ning Chien; Chu Chin Chen
Original assignee: Food Industry Res & Dev Inst
Priority date: 2012-09-06
Filing date: 2012-09-06
Publication date: 2014-10-21
Also published as: TW201410252A

Description

胜肽及其於抑制血管收縮素轉換酶之用途Peptide and its use for inhibiting angiotensin converting enzyme

本發明係有關可用於抑制血管收縮素轉換酶之新穎之胜肽。The present invention relates to novel peptides useful for inhibiting angiotensin converting enzyme.

血管收縮素轉換酶(Angiotensin Converting Enzyme,ACE)主要存在於人體的血管內皮細胞、肺部、腎臟及腦部。該酵素可將無活性的血管收縮素I上C-端的兩個胺基酸(His-Leu)移除，得到經活化的血管收縮素II，造成血管的收縮及血壓的上升(Maruyama,S.and Suzuki,H.,A peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein,Agric.Biol.Chem.,1982,46：1393-1394)。Maruyama,S.等人(Angiotensin I-converting enzyme inhibitor derived from an enzymatic hydrolysate of casein.II.Isolation and bradykinin-potentiating activity on the uterus and the ileum of rats,Agric.Biol.Chem.,1985；49：1405-1409)發現ACE可將血管舒張激肽(具有血管擴張活性)去活化，而造成血壓升高的現象。因此，血管收縮素轉換酶抑制劑(ACEI)與ACE的結合，可減少血管收縮素II的形成及血管舒張激肽之去活化，故ACEI的施用可改善高血壓的症狀。Angiotensin Converting Enzyme (ACE) is mainly found in human vascular endothelial cells, lungs, kidneys and brain. The enzyme removes the two amino acids (His-Leu) at the C-terminus of the inactive angiotensin I to obtain activated angiotensin II, causing blood vessel contraction and blood pressure rise (Maruyama, S. And Suzuki, H., A peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein, Agric. Biol. Chem., 1982, 46: 1393-1394). Maruyama, S. et al. (Angiotensin I-converting enzyme inhibitor derived from an enzymatic hydrolysate of casein. II. Isolation and bradykinin-potentiating activity on the uterus and the ileum of rats, Agric. Biol. Chem., 1985; 49:1405 -1409) It was found that ACE deactivates vasodilation kinin (having vasodilating activity), causing an increase in blood pressure. Therefore, the combination of angiotensin-converting enzyme inhibitor (ACEI) and ACE can reduce the formation of angiotensin II and deactivation of vasodilatation, so the administration of ACEI can improve the symptoms of hypertension.

Krysiak,R.等人(The effect of angiotensin-converting enzyme inhibitors on plasma adipokine levels in normotensive patients with coronary artery disease,Polish Journal of Endocrinology,2010,61：280-286)揭露ACE抑制劑對罹患冠狀動脈疾病(CAD)的病患為一種有效的治療劑，即使該等病患的血壓在正常的限制範圍內。美國專利第8,021,697 B2號揭示ACE抑制劑可藉由降低脂肪的整體百分率及/或增加瘦肉質量對脂肪質量的比例來改變身體質量的分布。換言之，ACE抑制劑可降低由膳食中所產生脂肪之量。Krysiak, R. et al. (The effect of angiotensin-converting enzyme inhibitors on plasma adipokine levels in normotensive patients with coronary artery disease, Polish Journal of Endocrinology, 2010, 61: 280-286) discloses that ACE inhibitors are an effective therapeutic agent for patients suffering from coronary artery disease (CAD), even if the blood pressure of such patients is within normal limits. U.S. Patent No. 8,021,697 B2 discloses that ACE inhibitors can alter the distribution of body mass by reducing the overall percentage of fat and/or increasing the ratio of lean mass to fat mass. In other words, ACE inhibitors can reduce the amount of fat produced by the diet.

除化學合成的藥物外，已發現多種具有不同長度及胺基酸殘基的胜肽可有效抑制ACE。該等具有ACE抑制活性的胜肽可自食品中分離獲得，該等食品包含，如動物或植物蛋白質的水解物，例如酪蛋白(Maruyama及Suzuki,1982；Maruyama,S.及Suzuki,H.,1985；及Yamamoto,N.等人.,Antihypertensive effect of peptides derived from casein by an extracellular proteinase fromLactobacillus helveticus CP790,J.Dairy Sci.,1994,77：917-922)、玉米蛋白(如α-玉米醇溶蛋白(α-zein))(Miyoshi等人，Structures and activity of angiotensin-converting enzyme inhibitors in an α-zein hydrolysate,Agric.Biol.Chem.,1991,55：1313-1318及Yano,S.等人，Isolation from α-zein of thermolysin peptides with angiotensin I-converting enzyme inhibitory activity,Biosci.Biotech.Biochem.,1996,60：661-663)、沙丁魚(Matsui,T.等人，Inhibition of angiotensin I-converting enzyme byBacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle,Biosci.Biotech.Biochem., 1993,57：922-925及Matsufuji,H.等人，Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle,Biosci.Biotech.Biochem.,1994,58：2244-2245)及鰹魚(Matsumura,N.等人，Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from bonito bowels,Biosci.Biotech.Biochem.,1993,57：1743-1744及Fujita,H.等人，Antihypertensive effect of thermolysin digest of dried bonito in spontaneously hypertensive rat,Clin.Exp.Pharmacol.Physiol.Suppl.,1995,22：S304-S305)；及發酵食品，例如清酒與酒渣(Saito,Y.等人，Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees,Biosci.Biotechnol.Biochem.,1994,58：1767-1771)、醬油(Kinoshita,E.等人，Purification and identification of an angiotensin I-converting enzyme inhibitor from soy sauce,Biosci.Biotechnol.Biochem.,1993,57：1107-1110)、乳酪(Okamoto,A.等人，Angiotensin I converting enzyme inhibitory activities of various fermented foods,Biosci.Biotechnol.Biochem.,1995,59：1147-1149)及酸乳(Masuda,O.等人，Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive rats,J.Nutr.,1996,126：3063-3068)。In addition to chemically synthesized drugs, a variety of peptides having different lengths and amino acid residues have been found to be effective in inhibiting ACE. Such peptides having ACE inhibitory activity can be isolated from foods, such as hydrolysates of animal or vegetable proteins, such as casein (Maruyama and Suzuki, 1982; Maruyama, S. and Suzuki, H., 1985; and Yamamoto, N. et al., Antihypertensive effect of peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP790, J. Dairy Sci., 1994, 77: 917-922), zein (eg alpha-cornol) Lysin (α-zein)) (Miyoshi et al., Structures and activity of angiotensin-converting enzyme inhibitors in an α-zein hydrolysate, Agric. Biol. Chem., 1991, 55: 1313-1318 and Yano, S. et al. , Isolation from α-zein of thermolysin peptides with angiotensin I-converting enzyme inhibitory activity, Biosci. Biotech. Biochem., 1996, 60: 661-663), sardines (Matsui, T. et al., Inhibition of angiotensin I-converting enzyme By Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle, Biosci. Biotech. Biochem., 1993, 57: 922-925 and Matsufuji, H. et al., Angiotensin I-converting enzyme i Nhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle, Biosci. Biotech. Biochem., 1994, 58: 2244-2245) and squid (Matsumura, N. et al., Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides) From bonito bowels, Biosci. Biotech. Biochem., 1993, 57: 1743-1744 and Fujita, H. et al., Antihypertensive effect of thermolysin digest of dried bonito in spontaneously hypertensive rat, Clin. Exp. Pharmacol. Physiol. Suppl., 1995, 22: S304-S305); and fermented foods such as sake and slag (Saito, Y. et al., Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees, Biosci. Biotechnol. Biochem., 1994 , 58: 1767-1771), soy sauce (Kinoshita, E. et al., Purification and identification of an angiotensin I-converting enzyme inhibitor from soy sauce, Biosci. Biotechnol. Biochem., 1993, 57: 1107-1110), cheese ( Okamoto, A. et al., Angiotensin I converting enzyme inhibitory activities of various fermented foods, Biosci. Biotechnol. Bioch Em., 1995, 59: 1147-1149) and yogurt (Masuda, O. et al., Antihypertetic peptides are present in aorta after oral administration of sour milk containing peptides to spontaneously hypertensive rats, J. Nutr., 1996, 126 :3063-3068).

日本專利特開第7289281(A)號揭示將大豆以黑麴菌(Aspergillus niger )發酵後的產物具有ACE抑制活性。日本專利特開第42999911(A)號揭示將大豆以鳳梨酶(bromelain；一種水解酶)水解後的胜肽產物具有ACE抑制活性。日本專利特開第2002053595(A)號揭示來自大豆水解物的胜肽可抑制ACE活性。日本專利特開第6298794(A)號揭示以源自動物及植物(如魚肉、豬肉及雞肉)的蛋白質來製備ACEI之方法。該水解物經離心、過濾、濃縮及樹脂吸附後，所獲得的胜肽具有ACE抑制活性。日本專利特開第5331192(A)號揭示將乾柴魚(katsuobushi)以嗜熱菌蛋白酶(thermolysin)水解後可產生具有ACE抑制活性的胜肽。日本專利特開第4264098(A)號揭示利用無脂肌肉製備具有ACE抑制活性胜肽的方法。美國專利第6,767,990 B1號揭示由雞殘渣水解物中分離出的之胜肽。美國公開第20120107409 A1揭示製備魚皮發酵產物之方法，該發酵產物可於抑制酪胺酸酶活性、抑制血管收縮素轉換酶活性及/或增進纖維母細胞之存活率。Japanese Patent Laid-Open No. 7287281 (A) discloses that the product obtained by fermenting soybean with Aspergillus niger has ACE inhibitory activity. Japanese Patent Laid-Open No. 42999911 (A) discloses that the peptide product obtained by hydrolyzing soybean with bromelain (a hydrolase) has ACE inhibitory activity. Japanese Patent Laid-Open No. 2002053595 (A) discloses that a peptide derived from a soybean hydrolyzate can inhibit ACE activity. Japanese Patent Laid-Open No. 6279794 (A) discloses a method for preparing ACEI from proteins derived from animals and plants such as fish, pork and chicken. After the hydrolyzate is centrifuged, filtered, concentrated, and adsorbed by a resin, the obtained peptide has ACE inhibitory activity. Japanese Patent Laid-Open No. 5331192 (A) discloses that katsuobushi can be hydrolyzed by thermolysin to produce a peptide having ACE inhibitory activity. Japanese Patent Laid-Open No. 4264098 (A) discloses a method of preparing a peptide having an ACE inhibitory activity using a fat-free muscle. U.S. Patent No. 6,767,990 B1 discloses a peptide isolated from a chicken residue hydrolysate. U.S. Patent No. 20,120,107, 409 A1 discloses a method of preparing a fish skin fermentation product which inhibits tyrosinase activity, inhibits angiotensin converting enzyme activity, and/or enhances fibroblast survival.

惟在此ACE抑制劑之領域中，仍需要開發特別是來自天然來源，且較化學合成化合物更安全的胜肽。However, in the field of ACE inhibitors, there is still a need to develop peptides that are particularly safer than chemically synthesized compounds, especially from natural sources.

本發明係關於可抑制ACE之新穎胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu- Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)及Trp-Asn。The present invention relates to novel peptides which inhibit ACE: Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val -Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu- Phe, Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3) and Trp-Asn.

本發明之另一態樣係關於一種組合物，其包含有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu-Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn、Phe-Val及Leu-Leu，及一或多種載劑、稀釋劑、溶劑、著色劑、抗氧化物、惰性物質及/或添加劑。Another aspect of the invention pertains to a composition comprising an effective amount of one or more peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly ( SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn- Ser-Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val and Leu-Leu, and one or more Agents, diluents, solvents, colorants, antioxidants, inerts and/or additives.

本發明之進一步態樣係提供用於預防及治療有此需要個體中一或多種與血管收縮素轉換酶有關之症狀的方法，其包含對該個體投與本發明之組合物或有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu-Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn、Phe-Val及Leu-Leu。A further aspect of the invention provides a method for the prevention and treatment of one or more symptoms associated with angiotensin converting enzyme in an individual in need thereof, comprising administering to the individual one of the compositions or effective amounts of the invention Or a plurality of peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn- His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys, Val -Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val and Leu-Leu.

本發明提供具有下列序列之經分離胜肽：Tyr-Tyr；Thr-Phe；Thr-Ser-Phe；Asn-Asp-Glu-Gly(SEQ ID NO：1)； Phe-Asp-Thr；Phe-Val-Asn-His-Phe(SEQ ID NO：2)；Gly-Leu-Phe；Val-Val-Asn；Thr-Tyr-Ala；Asn-Ser-Leu；Leu-Phe；Gly-Asn-Phe；Lys-Lys；Val-Gly-Gly-Ser(SEQ ID NO：3)；及Trp-Asn。The present invention provides an isolated peptide having the following sequence: Tyr-Tyr; Thr-Phe; Thr-Ser-Phe; Asn-Asp-Glu-Gly (SEQ ID NO: 1); Phe-Asp-Thr; Phe-Val-Asn-His-Phe (SEQ ID NO: 2); Gly-Leu-Phe; Val-Val-Asn; Thr-Tyr-Ala; Asn-Ser-Leu; Leu-Phe ; Gly-Asn-Phe; Lys-Lys; Val-Gly-Gly-Ser (SEQ ID NO: 3); and Trp-Asn.

此等胜肽可由天然物質(如動物(例如魚或雞)及植物(例如大豆)蛋白質)之水解物中分離並純化獲得。例如，該等蛋白質可根據美國專利第6,767,990 B1號、美國專利第8,021,697 B2號及美國公開第20120107409 A1所揭示之方法獲得，該等文獻的內容併入作為本案之部分。Such peptides can be obtained by isolating and purifying a hydrolysate of a natural substance such as an animal (for example, fish or chicken) and a plant (for example, soybean) protein. For example, such proteins can be obtained according to the methods disclosed in U.S. Patent No. 6,767,990 B1, U.S. Patent No. 8,021,697 B2, and U.S. Patent No. 20,120,107,409, the disclosure of which is incorporated herein by reference.

本發明具有ACE抑制活性之胜肽亦可以習知化學合成法製備。例如，氮疊化合物法、酸氯化物法、酸酐法、混合酸酐法、DCC法、活性酯法、碳基咪唑法、氧化還原法或DCC活化法(參見如Schroder與Luhke,The Peptide,Vol.1(1966),Academic Press,New York,USA出版，或Izumiya等人，Peptide synthesis,Maruzen Co.,Ltd(1975)出版，該等文獻的內容併入作為本案之部分。)。此等胜肽之合成可在固相或液相中進行。具有側鏈官能基如色胺酸及絲胺酸之胺基酸，較佳係將其側鏈官能基團，以已知之保護基團如苄氧羰基、第三丁氧基羰基或苄基等進行保護。此保護基團可再以任何習知之方法移除。The peptide having the ACE inhibitory activity of the present invention can also be prepared by a conventional chemical synthesis method. For example, nitrogen stack method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, carbon-based imidazole method, redox method or DCC activation method (see, for example, Schroder and Luhke, The Peptide, Vol. 1 (1966), Academic Press, New York, USA, or Izumiya et al., Peptide synthesis, Maruzen Co., Ltd (1975), the contents of which are incorporated herein by reference. The synthesis of these peptides can be carried out in a solid phase or in a liquid phase. An amino acid having a side chain functional group such as tryptophan and serine, preferably having a side chain functional group, a known protecting group The group is protected by a benzyloxycarbonyl group, a tert-butoxycarbonyl group or a benzyl group. This protecting group can then be removed by any conventional method.

或者，根據胺基酸序列，其亦可藉由將該胜肽之胺基酸序列之對應核苷酸序列選殖入適當的載體，再由適當的細胞、植物或動物表現。Alternatively, depending on the amino acid sequence, it may also be expressed by appropriate cells, plants or animals by colonizing the corresponding nucleotide sequence of the amino acid sequence of the peptide into an appropriate vector.

根據本發明，所謂「經分離」或「分離」意表該物質已由其原始環境(如其是天然存在之物質，意指天然環境)中移出。「經分離」並不意表其必然可擴張解釋為該物質中之所有其他物質(如雜質)已被移除而該物質已被純化。According to the present invention, "separated" or "isolated" means that the substance has been removed from its original environment (e.g., it is a naturally occurring substance, meaning a natural environment). "Separated" is not intended to mean that its inevitable expansion is interpreted as the removal of all other substances (such as impurities) in the substance that have been purified.

本發明之進一步態樣係提供一種組合物，其包含有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu-Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn、Phe-Val及Leu-Leu。於本發明之另一態樣中，該組合物包含有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu-Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn、Phe-Val及Leu-Leu，限制條件為Phe-Val及Leu-Leu不會同時存在。本發明之組合物係為醫藥組合物或食品組合物。本發明之醫藥組合物或食品組合物可藉由本發明所屬技術領域中所普遍知悉之操作方法製備，如將胜肽產物與一或多種習知之載劑、稀釋劑、溶劑、著色劑、抗氧化劑、惰性物質及/或添加劑將組合物調配成錠劑、膠囊、粉劑、粒劑、沖劑、濃縮劑、飲料、健康食品、食品添加劑或飼料。A further aspect of the invention provides a composition comprising an effective amount of one or more peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ) ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser -Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val and Leu-Leu. In another aspect of the invention, the composition comprises an effective amount of one or more peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ) ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser -Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val and Leu-Leu, with the restriction condition Phe-Val And Leu-Leu will not exist at the same time. The composition of the present invention is a pharmaceutical composition or a food composition. The pharmaceutical composition or food composition of the present invention can be Prepared by methods of operation generally known in the art to which the present invention pertains, such as blending a peptide product with one or more conventional carriers, diluents, solvents, colorants, antioxidants, inerts, and/or additives Tablets, capsules, powders, granules, granules, concentrates, beverages, health foods, food additives or feed.

根據本發明，本發明所提供的活性成分的「有效量」意表該成分的量足以達到所欲之ACE活性的抑制。該所需的正確量可依不同個體的狀況而改變，如該個體之疾病狀態、生理條件、年齡、性別、種族及體重；特定的個人及組合物的配方等。因此，並無法特別界定確實的「有效量」為何。然而，本技術領域中之技藝人士可使用例行的實驗測定適當的有效量。In accordance with the present invention, an "effective amount" of an active ingredient provided herein means that the amount of the ingredient is sufficient to achieve inhibition of the desired ACE activity. The correct amount required may vary depending on the condition of the individual, such as the disease state, physiological condition, age, sex, race and weight of the individual; the formulation of the particular individual and composition, and the like. Therefore, it is not possible to specifically define the exact "effective amount". However, one of ordinary skill in the art can use routine experimentation to determine an appropriate effective amount.

本發明之另一態樣係提供一種用於預防或治療有此需要個體中一或多種與血管收縮素轉換酶有關之症狀的方法，其包含對該個體投與本發明之組合物或有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu-Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn、Phe-Val及Leu-Leu。Another aspect of the present invention provides a method for preventing or treating one or more symptoms associated with angiotensin converting enzyme in an individual in need thereof, comprising administering to the individual a composition or an effective amount of the present invention One or more peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val- Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn, Phe-Val and Leu-Leu.

根據本發明，所謂「預防性(preventing)」或「預防(prevention)」意表當使用於有關病況時，包含在病況開始前向個體投與一藥劑後，相較於未接受該藥劑之個體，該經投藥之個體藥理病況的症狀之出現頻率或嚴重度會減緩，或開始之時間會延後。According to the present invention, the term "preventing" or "prevention" means, when used in a condition, includes administering a dose to an individual prior to the start of the condition, as compared to an individual not receiving the agent, The The frequency or severity of symptoms of the pharmacological condition of the individual being administered will be slowed down, or the time of initiation will be delayed.

根據本發明，所謂「治療性(treating)」或「治療(treatment)」意表當施用時，可回轉疾病或病況或其之一或多種症狀、減輕疾病或病況或其之一或多種症狀、抑制疾病或病況或其之一或多種症狀的發展，或者改善疾病或病況或其之一或多種症狀。According to the present invention, "treating" or "treatment" means, when administered, a disease or condition, or one or more of its symptoms, amelioration of a disease or condition, or one or more symptoms thereof, inhibition thereof. The development of a disease or condition, or one or more of its symptoms, or an improvement in a disease or condition, or one or more of its symptoms.

根據本發明，所謂「個體」意表任何動物，較佳為哺乳動物，且更佳為人類。個體的例子包括人類、非人靈長類、齧齒類、天竺鼠、兔子、羊、豬、山羊、牛、馬、狗及貓。According to the invention, the term "individual" means any animal, preferably a mammal, and more preferably a human. Examples of individuals include humans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cows, horses, dogs, and cats.

本發明技術領域之技藝人士瞭解ACE與如心血管併發症及脂肪細胞肥大及/或增殖等症狀有關。因此，本發明之胜肽及組合物具有治療或預防動脈性高血壓、心臟收縮性高血壓、周邊血管疾病、動脈粥樣硬化、再狹窄症、心臟衰竭、心功能不全、血栓形成及任何血栓栓塞症、心絞痛、腦血管意外事件、冠狀動脈疾病、心肌梗塞、血管再造及其組合，及\或減少腹部脂肪及/或皮下脂肪之功效。Those skilled in the art of the present invention recognize that ACE is associated with symptoms such as cardiovascular complications and adipocyte hypertrophy and/or proliferation. Accordingly, the peptides and compositions of the present invention have therapeutic or prophylactic arterial hypertension, systolic hypertension, peripheral vascular disease, atherosclerosis, restenosis, heart failure, cardiac insufficiency, thrombosis, and any thrombosis Embolism, angina pectoris, cerebrovascular accidents, coronary artery disease, myocardial infarction, revascularization and combinations thereof, and/or reduction of abdominal fat and/or subcutaneous fat.

下列實施例係以進一步說明本發明之可實施性，俾使本發明技術內容更加具體，然非欲以侷限本發明之範圍。其它熟習該項技術者基於習知技藝之教示而可達成的本發明之種種變化及改良，皆應歸屬本發明範疇。The following examples are intended to further illustrate the exemplification of the present invention, and the technical scope of the present invention is more specific, and is not intended to limit the scope of the invention. Various changes and modifications of the present invention which can be made by those skilled in the art based on the teachings of the art are intended to fall within the scope of the present invention.

實施例Example 實施例1大豆殘渣之製備Example 1 Preparation of Soybean Residue

於25公斤之脫脂大豆粉(購自中聯油脂公司)中加入該脫脂大豆粉一半重量的水，並將其混合。將該混合物於100℃下蒸煮45分鐘後冷卻至45℃。將4.2克含Aspergillus sojae 之麴粉(購自日本樋口公司)加入該混合物中並混合均勻，且於相對濕度95%、溫度27℃下發酵48小時。於此發酵產物中加入發酵產物三倍重量的水，再將混合物於45℃下水解8小時。以濾布使水解物中之液體部分與大豆殘渣分離。To 25 kg of defatted soy flour (purchased from Zhonglian Oil Co., Ltd.), half of the weight of the defatted soy flour was added and mixed. The mixture was cooked at 100 ° C for 45 minutes and then cooled to 45 ° C. 4.2 g of Aspergillus sojae (purchased from Japan's Sakaguchi Co., Ltd.) was added to the mixture and uniformly mixed, and fermented at a relative humidity of 95% and a temperature of 27 ° C for 48 hours. Three times by weight of water of the fermentation product was added to the fermentation product, and the mixture was further hydrolyzed at 45 ° C for 8 hours. The liquid portion of the hydrolyzate was separated from the soybean residue by a filter cloth.

實施例2具有ACE抑制活性胜肽之製備Example 2 Preparation of peptide with ACE inhibitory activity

將實例1中所得之大豆殘渣與1 L的水混合均勻，並調整混合物之pH值至2.0。將0.5 g的胃蛋白酶加入混合物中，並使其在37℃下水解2小時獲得水解物。以NaHCO₃ 將水解物的之pH值調整至7.2。將0.5 g胰蛋白酶及0.5 g胰凝乳蛋白酶加入該水解物中，並使其在37℃下進一步水解2.5小時以獲得一進一步水解物。將該進一步水解物以沸水浴加熱15分鐘以使酵素去活化。冷卻後，將該進一步水解物在10,000 rpm下離心10分鐘，以使上清液與固體沉澱物分離。將該上清液冷凍乾燥而獲得43 g之乾粉末，其中蛋白質的含量為24.5%。The soybean residue obtained in Example 1 was uniformly mixed with 1 L of water, and the pH of the mixture was adjusted to 2.0. 0.5 g of pepsin was added to the mixture and allowed to hydrolyze at 37 ° C for 2 hours to obtain a hydrolyzate. The pH of the hydrolyzate was adjusted to 7.2 with NaHCO ₃ . 0.5 g of trypsin and 0.5 g of chymotrypsin were added to the hydrolyzate, and further hydrolyzed at 37 ° C for 2.5 hours to obtain a further hydrolyzate. The further hydrolyzate was heated in a boiling water bath for 15 minutes to deactivate the enzyme. After cooling, the further hydrolyzate was centrifuged at 10,000 rpm for 10 minutes to separate the supernatant from the solid precipitate. The supernatant was freeze-dried to obtain 43 g of a dry powder having a protein content of 24.5%.

將乾粉末溶於水中以配製成1%(w/v)之水溶液。將該水溶液以孔徑為0.45 μm的薄膜過濾。將濾液以高效率液相層析質譜儀(HPLC)進行分析。所使用尺寸篩除管柱(size exclusion column)的條件如下：系統：AKTA純化器(purifier)The dry powder was dissolved in water to prepare a 1% (w/v) aqueous solution. The aqueous solution was filtered through a membrane having a pore size of 0.45 μm. The filtrate was analyzed by high performance liquid chromatography mass spectrometry (HPLC). The conditions used to screen the size exclusion column are as follows: System: AKTA purifier

沖離管柱：Superdex Peptide HR 10/30Flushing the column: Superdex Peptide HR 10/30

樣品量：500 μLSample size: 500 μL

溶離液：5%醇溶於純水Dissolved solution: 5% alcohol dissolved in pure water

流速：0.25 mL/minFlow rate: 0.25 mL/min

檢出器：214 nm。Detector: 214 nm.

每20分鐘收取5 mL由管柱中沖離出的樣本，並總共收取7個沖離餾份。上述HPLC程序重複60次，將所有收取的對應餾份分別混合在一起，並經凍乾後獲得7個乾粉末樣本。再將各乾粉末樣本溶於水中以製備1%(w/v)之水溶液。所有水溶液之ACE抑制活性係個別以Vermeirssen,V.等人(Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides,J.Biochem.Biophys.Methods,2002,51：75-87)所揭示之方法測量。如表1所示，於所有溶液中，餾份3、4及5之溶液的ACE抑制活性較強，且以餾份3之溶液的活性最強。5 mL of sample flushed out of the column was taken every 20 minutes and a total of 7 flushed fractions were charged. The above HPLC procedure was repeated 60 times, and all the corresponding fractions collected were separately mixed together and lyophilized to obtain 7 dry powder samples. Each dry powder sample was dissolved in water to prepare a 1% (w/v) aqueous solution. The ACE inhibitory activity of all aqueous solutions is individually Vergilssen, V. et al. (Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides, J. Biochem. Biophys. Methods, 2002, 51: 75-87). The method disclosed is measured. As shown in Table 1, among all the solutions, the solutions of the fractions 3, 4 and 5 had a strong ACE inhibitory activity, and the solution of the fraction 3 was the most active.

將第3餾份之溶液再以Sephasil Peptide RP-18管柱，依下列條件沖離：系統：AKTA純化器The solution of the third fraction was further separated on a Sephasil Peptide RP-18 column by the following conditions: System: AKTA Purifier

沖離管柱：Sephasil Peptide RP-18Flushing the column: Sephasil Peptide RP-18

樣品量：100 μLSample size: 100 μL

溶離液A：含有0.1%三氟乙酸的H₂ ODissolution A: H ₂ O containing 0.1% trifluoroacetic acid

溶離液B：含有0.1%三氟乙酸的MeOHDissolution B: MeOH containing 0.1% trifluoroacetic acid

梯度：0%的B為時5minGradient: 0% of B is 5 min

0-100%的B為時30 min0-100% of B is 30 min

100%的B為時10 min100% of B is 10 min

流速：1 mL/minFlow rate: 1 mL/min

檢出器：214 nm。Detector: 214 nm.

每5分鐘收取由管柱中沖離出的樣本，並總共收取6個沖離餾份。上述沖離程序重複250次。將所有的6個沖離餾份分別混合在一起，並經凍乾後獲得6個乾粉末樣本。再將各乾粉末樣本溶於1 mL水中以製備水溶液。測量各餾份之水溶液之ACE抑制活性。其結果顯示，餾份3-2、3-3及3-4之抑制活性較佳，且以餾份3-4之抑制活性最佳(見下示之表2)。Samples rushed out of the column were taken every 5 minutes and a total of 6 effluent fractions were charged. The above flushing procedure was repeated 250 times. All 6 rinse-off fractions were mixed together and lyophilized to obtain 6 dry powder samples. Each dry powder sample was dissolved in 1 mL of water to prepare an aqueous solution. The ACE inhibitory activity of the aqueous solution of each fraction was measured. As a result, it was found that the inhibitory activities of the fractions 3-2, 3-3 and 3-4 were better, and the inhibitory activity of the fractions 3-4 was the best (see Table 2 shown below).

將餾份3-4經Luna C18(2)管柱以下列條件沖離後，再以質譜儀檢測m/z 100-800之物質：泵：Water 600Distillate 3-4 was pulverized through a Luna C18 (2) column under the following conditions, and then the mass spectrometer was used to detect the substance of m/z 100-800: pump: Water 600

沖離管柱：Luna C18(2)150^＊ 2 mm,3 μFlushing the column: Luna C18(2)150 ^* 2 mm, 3 μ

樣品量：20 μLSample size: 20 μL

溶離液A：含有0.1%甲酸的H₂ ODissolution A: H ₂ O containing 0.1% formic acid

溶離液B：含有0.1%甲酸的乙腈Dissolution B: Acetonitrile containing 0.1% formic acid

梯度：5%的B為時5 minGradient: 5% of B is 5 min

5-95%的B為時24 min5-95% of B is 24 min

95%的B為時10 min95% of B is 10 min

流速：0.2 mL/minFlow rate: 0.2 mL/min

檢出器：Quattro LC MS/MS。Detector: Quattro LC MS/MS.

結果鑑定出17個胜肽。根據Pripp,A.H.等人(Quantitative structure-activity relationship modeling of ACE-inhibitory peptides derived from milk proteins,Eur.Food Res.Technol.,2004,219：579-583)所揭示的方法計算該17個胜肽的ACE抑制活性。根據下示表3所示的結果可發現，所有的該17個胜肽皆可有效地抑制ACE之活性，且因此可用作ACE抑制劑。As a result, 17 peptides were identified. The 17 wins were calculated according to the method disclosed by Quantitative structure-activity relationship modeling of ACE-inhibitory peptides derived from milk proteins, Eur. Food Res. Technol., 2004, 219: 579-583. The ACE inhibitory activity of the peptide. According to the results shown in Table 3 below, it was found that all of the 17 peptides were effective in inhibiting the activity of ACE, and thus were useful as ACE inhibitors.

<110> 財團法人食品工業發展研究所<110> Food Industry Development Research Institute

<120> 貹肽及其於抑制血管收縮素轉換酶之用途<120> 貹 peptide and its use for inhibiting angiotensin converting enzyme

<130> US2429<130> US2429

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<170> PatentIn version 3.5<170> PatentIn version 3.5

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<213> 人工序列<213> Artificial sequence

<220><220>

<223> 合成肽<223> Synthetic peptide

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<211> 5<211> 5

<212> PRT<212> PRT

<213> 人工序列<213> Artificial sequence

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<223> 合成肽<223> Synthetic peptide

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<213> 人工序列<213> Artificial sequence

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<223> 合成肽<223> Synthetic peptide

<400> 3 <400> 3

Claims

一種經分離之胜肽，其係選自下列所組成之群：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)及Trp-Asn。 An isolated peptide selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp- Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Gly-Asn-Phe, Lys-Lys, Val-Gly -Gly-Ser (SEQ ID NO: 3) and Trp-Asn.

一種用於預防或治療一或多種與血管收縮素轉換酶有關之症狀的醫藥組合物，其包含有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn及Phe-Val，及一或多種載劑、稀釋劑、溶劑、著色劑、抗氧化物、惰性物質及/或添加劑。 A pharmaceutical composition for preventing or treating one or more symptoms associated with an angiotensin converting enzyme comprising an effective amount of one or more peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser- Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Val-Val-Asn, Thr-Tyr- Ala, Asn-Ser-Leu, Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn and Phe-Val, and one or more carriers, diluents , solvents, colorants, antioxidants, inerts and/or additives.

如請求項2之醫藥組合物，其中該症狀為心血管併發症。 The pharmaceutical composition of claim 2, wherein the symptom is a cardiovascular complication.

如請求項3之醫藥組合物，其中該心血管併發症係選自動脈性高血壓、心臟收縮性高血壓、周邊血管疾病、動脈粥樣硬化、再狹窄症、心臟衰竭、心功能不全、血栓形成及任何血栓栓塞症、心絞痛、腦血管意外事件、冠狀動脈疾病、心肌梗塞、血管再造及其組合。 The pharmaceutical composition according to claim 3, wherein the cardiovascular complication is selected from the group consisting of arterial hypertension, systolic hypertension, peripheral vascular disease, atherosclerosis, restenosis, heart failure, cardiac insufficiency, thrombosis Formation and any thromboembolism, angina pectoris, cerebrovascular accident, coronary artery disease, myocardial infarction, revascularization, and combinations thereof.

如請求項2之醫藥組合物，其中該症狀為脂肪細胞肥大及/或增殖。 The pharmaceutical composition of claim 2, wherein the symptom is fat cell hypertrophy And / or proliferation.

如請求項2或5之醫藥組合物，其係用於減少腹部脂肪及/或皮下脂肪。 A pharmaceutical composition according to claim 2 or 5 for use in reducing abdominal fat and/or subcutaneous fat.

一種用於預防或治療一或多種與血管收縮素轉換酶有關之症狀的食品組合物，其包含有效量之一或多種選自下列之胜肽：Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn及Phe-Val，及一或多種載劑、稀釋劑、溶劑、著色劑、抗氧化物、惰性物質及/或添加劑。 A food composition for preventing or treating one or more symptoms associated with angiotensin converting enzyme, comprising an effective amount of one or more peptides selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser- Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Val-Val-Asn, Thr-Tyr- Ala, Asn-Ser-Leu, Gly-Asn-Phe, Lys-Lys, Val-Gly-Gly-Ser (SEQ ID NO: 3), Trp-Asn and Phe-Val, and one or more carriers, diluents , solvents, colorants, antioxidants, inerts and/or additives.

如請求項7之食品組合物，其係為健康食品或食品添加劑。 A food composition according to claim 7 which is a health food or a food additive.

如請求項7或8之食品組合物，其中該症狀為心血管併發症。 The food composition of claim 7 or 8, wherein the symptom is a cardiovascular complication.

如請求項9之食品組合物，其中該心血管併發症係選自動脈性高血壓、心臟收縮性高血壓、周邊血管疾病、動脈粥樣硬化、再狹窄症、心臟衰竭、心功能不全、血栓形成及任何血栓栓塞症、心絞痛、腦血管意外事件、冠狀動脈疾病、心肌梗塞、血管再造及其組合。 The food composition of claim 9, wherein the cardiovascular complication is selected from the group consisting of arterial hypertension, systolic hypertension, peripheral vascular disease, atherosclerosis, restenosis, heart failure, cardiac insufficiency, thrombosis Formation and any thromboembolism, angina pectoris, cerebrovascular accident, coronary artery disease, myocardial infarction, revascularization, and combinations thereof.

如請求項7之食品組合物，其中該症狀為脂肪細胞肥大及/或增殖。 The food composition of claim 7, wherein the symptom is fat cell hypertrophy and/or proliferation.

如請求項7或11之食品組合物，其係用於減少腹部脂肪及/或皮下脂肪。 A food composition according to claim 7 or 11, which is for reducing belly fat And / or subcutaneous fat.

一種一或多種選自Tyr-Tyr、Thr-Phe、Thr-Ser-Phe、Asn-Asp-Glu-Gly(SEQ ID NO：1)、Phe-Asp-Thr、Phe-Val-Asn-His-Phe(SEQ ID NO：2)、Gly-Leu-Phe、Val-Val-Asn、Thr-Tyr-Ala、Asn-Ser-Leu、Leu-Phe、Gly-Asn-Phe、Lys-Lys、Val-Gly-Gly-Ser(SEQ ID NO：3)、Trp-Asn及Phe-Val的胜肽之用途，其係用以製備用於預防或治療一或多種與血管收縮素轉換酶有關之症狀的藥物。 One or more selected from the group consisting of Tyr-Tyr, Thr-Phe, Thr-Ser-Phe, Asn-Asp-Glu-Gly (SEQ ID NO: 1), Phe-Asp-Thr, Phe-Val-Asn-His-Phe (SEQ ID NO: 2), Gly-Leu-Phe, Val-Val-Asn, Thr-Tyr-Ala, Asn-Ser-Leu, Leu-Phe, Gly-Asn-Phe, Lys-Lys, Val-Gly- The use of peptides of Gly-Ser (SEQ ID NO: 3), Trp-Asn and Phe-Val for the preparation of a medicament for the prevention or treatment of one or more symptoms associated with angiotensin converting enzyme.

如請求項13之用途，其中該症狀為心血管併發症。 The use of claim 13, wherein the symptom is a cardiovascular complication.

如請求項14之用途，其中該心血管併發症係選自動脈性高血壓、心臟收縮性高血壓、周邊血管疾病、動脈粥樣硬化、再狹窄症、心臟衰竭、心功能不全、血栓形成及任何血栓栓塞症、心絞痛、腦血管意外事件、冠狀動脈疾病、心肌梗塞、血管再造及其組合。 The use of claim 14, wherein the cardiovascular complication is selected from the group consisting of arterial hypertension, systolic hypertension, peripheral vascular disease, atherosclerosis, restenosis, heart failure, cardiac insufficiency, thrombosis, and Any thromboembolic disorder, angina pectoris, cerebrovascular accident, coronary artery disease, myocardial infarction, revascularization, and combinations thereof.

如請求項13之用途，其中該症狀為脂肪細胞肥大及/或增殖。 The use of claim 13, wherein the symptom is adipose cell hypertrophy and/or proliferation.

如請求項13或16之用途，其係用於減少腹部脂肪及/或皮下脂肪。The use of claim 13 or 16 is for reducing abdominal fat and/or subcutaneous fat.